This application requests continued funding for our project to investigate the role of acid ceramidase (AC) in mammalian development and disease pathogenesis. During the previous funding period we have: a) shown that AC is essential for embryo survival beyond the 2-cell stage, b) demonstrated that AC is a critical component of oocyte development, and can be used to prevent oocyte and embryo apoptosis in vitro, c) revealed a novel, autocatalytic mechanism of AC processing and activation, and identified a new class of AC inhibitors, and d) constructed floxed AC conditional knockout mice carrying an inducible Cre recombinase (ACcKO/CreTM), and shown that viable ACKO-/- mice can be produced from these animals following tamoxifen injection. In the upcoming funding period we will extend these findings by pursuing the following two, interrelated aims: 1) Construct & characterize the first viable mouse models of AC deficiency. We will continue to use the ACcKO/CreTM mice to induce the ubiquitous knockout of AC activity at different stages of embryogenesis and after birth. We will also breed ACcKO mice to cell-specific (macrophage and Purkinje cell) Cre mice, and use these animals to evaluate the cell-specific functions of AC. The resulting AC deficient mice and embryos will be characterized pathologically, biochemically, and (for those that survive) clinically. In the event that the mice we obtain from these experiments do not have a phenotype that is clinically relevant to human AC deficiency (Farber disease), we will also create hypomorph mice using a mutation knock-in strategy. The goal of these studies is to gain further insights into the role of AC in mammalian development and disease pathogenesis, and to provide researchers with the first viable models of AC deficiency. 2) Further explore the in vivo role of AC in oocyte development. Oocytes are an excellent model for the study of mammalian apoptosis, and our work during the previous funding period has shown that AC is critical to this process. To pursue these findings further, three different Cre mice (GDF9-Cre, ZP3-Cre and Msx2-Cre) will be used to inactivate AC at different stages of folliculogenesis after breeding to the ACcKO mice produced in aim 1. Follicle morphology, as well as the number of oocytes retrieved following superovulation and their stage of development (GV, MI, MII), fertilization capacity, and apoptosis rates will be determined and compared. We will also use TM to inactivate AC in ovarian tissue, and study the uptake and trafficking of recombinant AC by mature oocytes. The goal of these studies is to gain a more complete understanding of AC's role in oocyte development, and to provide a deeper mechanistic appreciation of how AC influences in vitro maturation and fertilization. PUBLIC HEALTH RELEVANCE: AC is a key enzyme in the regulation of sphingolipid metabolism and signaling, and abnormal AC expression has been observed in many human diseases. Our previous work has provided many of the essential research tools needed to study this enzyme, and revealed its critical role in early development. The research we are proposing in the upcoming funding period will continue to provide important new information about this protein, and should impact a wide range of translational medicine disciplines (e.g., lysosomal disease research, fertility, sphingolipid signaling, etc).